SUMMARY Disease relapse is a leading cause of treatment failure after autologous (auto) and allogeneic (allo) hematopoietic stem cell transplantation (HCT) for patients with non-Hodgkin lymphoma (NHL). Accordingly, there is intense interest in using adoptive cellular immunotherapy to eradicate the limited number of tumor cells surviving after conditioning chemo-radiotherapy. For patients unable to undergo HCT or having relapsed post- HCT, this strategy may offer an option with curative intent. Chimeric Antigen Receptor (CAR)-engineered T cells have emerged as a promising treatment for relapsed/refractory CD19+ malignancies, but the full potential of this therapy is hampered by attenuated engraftment and lack of long-term persistence of CAR redirected T cells in patients. We propose a novel approach to improve efficacy and durability of CAR T cells based on properties of cytomegalovirus (CMV)-specific T cells and their expansion using a CMV vaccine, Triplex, recently developed and clinically evaluated at City of Hope. Triplex is a multi-antigen recombinant modified vaccinia Ankara (MVA) virus with genes encoding 3 CMV proteins, pp65, IE1, and IE2. CMV-MVA Triplex has proven safe and powerfully immunogenic in both CMV-seronegative and -seropositive healthy volunteers in a Phase I trial, and is now undergoing testing in alloHCT recipients. Our approach entails selecting CMVpp65- specific T cells for ex vivo modification with a CD19-targeting CAR, infusing the bi-specific CMV-CD19 CAR T cells into patients, and then inducing in vivo expansion by stimulating the native CMV-specific T cell receptor (TCR) using Triplex injections. Our strategy will shorten CAR T cell manufacturing time, with the dual advantage of preventing premature senescence of the infused cell product, and of making the product more rapidly available. Also, the truncated epidermal growth factor receptor (EGFRt) in our CAR design will act as both a tracking marker and an in vivo safety switch for ablating the CAR T cells. The proposed strategy is designed to enhance proliferation, lengthen persistence and augment the anti-lymphoma activity of adoptively transferred CMV-CD19 CAR T cells by re-stimulating these cells through the native CMVpp65-specific TCR. This should result in improved progression-free survival for NHL patients. The innovative aspect of this proposal is the substitution of in vitro expansion of the infused cell product with a novel in vivo expansion strategy. In Specific Aim 1 (SA1) we will validate this bi-specific CMV-CD19 CAR T cell manufacturing platform under cGMP conditions to produce clinical grade and scale cell products. In Specific Aims 2 and 3, we will test the CMV-CD19 CAR T cells therapy followed by Triplex vaccine in pilot studies for patients with relapsed- refractory B-cell NHL following autoHCT or lymphodepletion (SA2), or alloHCT (SA3). The significance of this approach is that we will have in vivo control of adoptively infused CMV-CD19 CAR T cell expansion and ablation. This project serves as a proof of principle for a method of enhancing CAR effectiveness and controlling T cell expansion that can potentially be applied to multiple diseases in multiple therapeutic settings.